Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution

Protein engineering techniques were used to construct a derivative of the serine protease subtilisin that ligates peptides efficiently in water. The subtilisin double mutant in which the catalytic Ser221 was converted to Cys (S221C) and Pro225 converted to Ala (P225A) has 10-fold higher peptide ligase activity and at least 100-fold lower amidase activity than the singly mutated thiolsubtilisin (S221C) that was previously shown to have some peptide ligase activity [Nakatsuka, T., Sasaki, T., & Kaiser, E.T. (1987) J. Am. Chem. Soc. 109, 3808-3810]. A 1.5-A X-ray crystal structure of an oxidized derivative of the double mutant (S221C/P225A) supports the protein design strategy in showing that the P225A mutation partly relieves the steric crowding expected from the S221C substitution, thus accounting for its improved catalytic efficiency. Stable and synthetically reasonable alkyl ester peptide substrates were prepared that rapidly acylate the S221C/P225A enzyme, and aminolysis of the resulting thioacyl-enzyme intermediate by various peptides is strongly preferred over hydrolysis. The efficiency of aminolysis is relatively insensitive to the sequence of the first two residues in the acyl acceptor peptide whose alpha-amino group attacks the thioacyl-enzyme. To obtain greater flexibility in the choice of coupling sites, a set of three additional peptide ligases were engineered by introducing mutations into the parent ligase (S221C/P225A) that were previously shown to change the specificity of subtilisin for the residue nearest the acyl bond (the P1 residue). The specificity properties of the parent ligase and derivatives of it paralleled those of wild type and corresponding specificity variants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the binding of plasminogen to fibrin surfaces: the role of carboxy-terminal lysines.

In the present study we have quantitatively characterized the interaction of purified human Glu- and Lys-plasminogen with intact and degraded fibrin by ligand-binding experiments using a radioisotopic dilution method and antibodies against human plasminogen. A fibrinogen monolayer was covalently linked to a solid support with polyglutaraldehyde and was treated with thrombin or with thrombin and then plasmin to respectively obtain intact and degraded fibrin surfaces. Under these conditions, a well-defined surface of fibrin is obtained (410 +/- 4 fmol/cm2) and, except for a 39-kDa fragment, most of the fibrin degradation products remain bound to the support. New binding sites for plasminogen were detected on the degraded surface of fibrin. These sites were identified as carboxy-terminal lysine residues both by inhibition of the binding by the lysine analogue 6-aminohexanoic acid and by carboxy-terminal end-group digestion with carboxypeptidase B. The binding curves exhibited a characteristic Langmuir adsorption isotherm saturation profile. The data were therefore analyzed accordingly, assuming a single-site binding model to simplify the analysis. Equilibrium dissociation constants (Kd) and the maximum number of binding sites (Bmax) were derived from linearized expression of the Langmuir isotherm equation. The Kd for the binding of Glu-plasminogen to intact fibrin was 0.99 +/- 0.17 microM and for degraded fibrin was 0.66 +/- 0.22 microM. The Kd for the binding of Lys-plasminogen to intact fibrin was 0.41 +/- 0.22 microM and for degraded fibrin was 0.51 +/- 0.12 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a major brain tubulin variant which cannot be tyrosinated

Brain tubulin preparations contain an abundant type of tubulin which does not undergo the normal cycle of tyrosination-detyrosination, and whose nature is still unknown. We have used peptide sequence analysis and mass spectrometry combined with immunological procedures to show that this non-tyrosinatable tubulin has a specific primary structure. It differs from the tyrosinated isotype in that it lacks a carboxy-terminal glutamyl-tyrosine group on its alpha-subunit. Thus, non-tyrosinatable tubulin originates from a well-defined posttranslational modification of the tubulin primary structure which is located at the expected site of activity of tubulin tyrosine ligase. This probably accounts for the reason why it cannot be tyrosinated. The significance of this abundant brain isotubulin and the metabolic pathway involved in its formation remain to be elucidated. This should shed light on the relation between the structural diversity of the carboxy terminus of alpha-tubulin and the regulation of functional properties of microtubules.     

The antimycin-A-insensitive respiratory pathway of Candida parapsilosis: evidence for a second quinone involved specifically in its functioning

The involvement of a quinone in the antimycin A-insensitive electron transfer from NADH-dehydrogenase to cytochrome c via the alternative respiratory chain of Candida parapsilosis, by-passing complex II, has been studied. After a partial extraction of quinones, the residual respiration was fully antimycin-A-sensitive, but reincorporation of the organic extract partially restored an antimycin A-insensitive respiration. Analysis of quinone content by HPLC, after purification by thin-layer chromatography, evidenced another quinone species in a very low amount. Myxothiazol and stigmatellin were shown to inhibit the alternative pathway but at a higher concentration than required to inhibit the classical pathway. Cytochrome spectra analysis showed that, in the presence of high myxothiazol concentrations, cytochromes c and aa3 were not reduced, while they were in the presence of antimycin A. It is suggested that the secondary pathway of C. parapsilosis involved a specific quinone pool which can be displaced from its binding site by high concentrations of myxothiazol or analogous compounds.     

Substrate specificity of alpha-chymotrypsin-catalyzed esterification in organic media.

11 amino acid derivatives were tested as alpha-chymotrypsin substrates in the esterification reaction with methanol in organic media. The reactions were carried out in water-saturated ethyl acetate and in acetonitrile containing 4% water. alpha-Chymotrypsin adsorbed on Celite was used as a catalyst. From initial reaction rate measurements, the Michaelis-Menten parameters Vmax and KM were determined. All the amino acid derivatives tested were esterified, and the highest values of kcat/KM were obtained with the N-acylated aromatic amino acids. Correlations between Michaelis-Menten parameters and physical properties of the substrates such as molar refractivity (MR) and log P were deduced. The results show that the specificity of the alpha-chymotrypsin towards the side chain of the amino acids in organic media is the same as that in aqueous media. However, the specificity towards the N-protecting group is opposite to that in water, so the reaction medium affects the interaction of this part of the molecule with the enzyme to a large extent.     

Cytidylyltransferase translocation onto endoplasmic reticulum and increased de novo synthesis without phosphatidylcholine accumulation in Krebs-II ascite cells.

Addition of oleic acid to Krebs-II cells stimulated by 9-fold [3H]choline incorporation into choline glycerophospholipids without affecting the selective incorporation of the precursor into diacyl subclass (90% of total [3H]choline glycerophospholipids). The total activity of cytidylyltransferase (E.C. 2.7.7.15), the regulatory enzyme of choline glycerophospholipid synthesis, was increased in the particulate fraction at the expense of cytosol. Free [3H]oleic acid was also associated with the particulate fraction. Subcellular fractionation of membranes on Percoll gradient, indicated that the endoplasmic reticulum, which contained 90% of total cell free oleic acid, was the unique target for the translocation of cytidylyltransferase. [3H]oleic acid was incorporated almost exclusively into phosphatidylcholine and corresponded to a synthesis of 9 nmol/h per 10(6) cells. Based on [3H]choline incorporation a net synthesis of 22 nmol/h per 10(6) cells was determined. However, oleic acid treatment did not change the total amount of phosphatidylcholine (45 nmol/10(6) cells) and other phospholipids; also no modification in the subcellular distribution of phospholipids was observed. It is concluded that the stimulation of the de novo synthesis of phosphatidylcholine which involves translocation of cytidylyltransferase onto the endoplasmic reticulum, is accompanied by a renewal of their polar head group. Also exogenous oleic acid induces an enhanced fatty acid turnover, highly specific for phosphatidylcholine. Therefore, Krebs-II cells exhibited a high degree of regulation of their phosphatidylcholine content, suggesting a parallel stimulation of both synthesis and degradation.     

Phosphorylation of Na,K-ATPase by acetyl phosphate and inorganic phosphate. Sidedness of Na+, K+ and nucleotide interactions and related enzyme conformations.

The effects of K+, Na+ and nucleotides (ATP or ADP) on the steady-state phosphorylation from [32P]Pi (0.5 and 1 mM) and acetyl [32P]phosphate (AcP) (5 mM) were studied in membrane fragments and in proteoliposomes with partially purified pig kidney Na,K-ATPase incorporated. The experiments were carried out at 20 degrees C and pH 7.0. In broken membranes, the Pi-induced phosphoenzyme levels were reduced to 40% by 10 mM K+ and to 20% by 10 mM K+ plus 1 mM ADP (or ATP); in the presence of 50 mM Na+, no E-P formation was detected. On the other hand, with AcP, the E-P formation was reduced by 10 mM K+ but was 30% increased by 50 mM Na+. In proteoliposomes E-P formation from Pi was (i) not influenced by 5-10 mM K+cyt or 100 mM Na+ext, (ii) about 50% reduced by 5, 10 or 100 mM K+ext and (iii) completely prevented by 50 mM Na+cyt. Enzyme phosphorylation from AcP was 30% increased by 10 mM K+cyt or 50 mM Na+cyt; these E-P were 50% reduced by 10-100 mM K+ext. However, E-P formed from AcP without K+cyt or Na+cyt was not affected by extracellular K+. Fluorescence changes of fluorescein isothiocyanate labelled membrane fragments, indicated that E-P from AcP corresponded to an E2 state in the presence of 10 mM Na+ or 2 mM K+ but to an E1 state in the absence of both cations. With pNPP, the data indicated an E1 state in the absence of Na+ and K+ and also in the presence of 20 mM Na+, and an E2 form in the presence of 5 mM K+. These results suggest that, although with some similarities, the reversible Pi phosphorylation and the phosphatase activity of the Na,K-ATPase do not share the whole reaction pathway.     

Vitamin K-dependent carboxylation of poly-L-glutamate.

Poly-L-glutamate preparations of varying chain length were used as substrates for bovine liver vitamin K-dependent carboxylase. The quality of these substrates (as measured by their apparent kinetic constants) was comparable to that of the more commonly used tri- and pentapeptides, but the maximal reaction rate increased at increasing chain length.     

Influence of pulsatility on the development of intracardiac jets: an in vitro laser Doppler study.

So far, it has been hypothesized that numerical data obtained in steady flow conditions apply to pulsatile flows. In order to study the modifications of the velocity fields due to pulsatility, jets were produced by 8 orifices (with a diameter "D" of 4.4 to 11.3 mm) included in a chamber of 50 mm. The velocity was measured using laser Doppler anemometry with a pulsatile flow ("pf") and compared to the values obtained in steady ("sf"): at maximum velocity, the longitudinal velocity profile is qualitatively similar to this observed in steady flow: it is made of a plateau followed by an hyperbolic velocity decay in the turbulent area. The length of the core ("Lpf") is strongly related to "D" (Lpf = 3.72 D + 5.49, r = .99) and the velocity decay depends on the ratio between the distance "x" from the orifice and "D" (V/Vo = 2.83D/x + 3.46, r = .85, where V is the velocity at "x" and Vo the initial velocity). During the acceleration and the deceleration, the laminar core is disturbed by turbulences. The comparison of "pf" data with "sf" data demonstrated similar diameters at the origin of the jets (Dpf = 0.96 Dsf + .12, r = .99), but significant (p less than .0001) differences both for "L" and "V/Vo": Lpf = .91Lsf + 6.58, r = .97, V/Vopf = .63 V/Vosf + .34, r = .76. Thus, pulsatility modifies velocity fields and the results obtained in steady flow conditions do not apply to pulsatile jets.